Current research from this laboratory indicates that synthetic peptides can act as substrates or inhibitors of cyclic nucleotide-dependent protein kinases. It appears that distinct differences exist in the determinants of substrate specificity required by the cyclic AMP-dependent and cyclic GMP-dependent kinases. The overall purpose of this proposal is to develop selective inhibitors of each enzyme. In the first section, peptides that are analogs of amino acid sequences around sites of phosphorylation in protein substrates will be synthesized. The potency and selectivity of these peptides as inhibitors of purified protein kinases will be kinetically characterized. The peptide inhibitors will be evaluated by determining the type of inhibition exhibited, the relative inhibition constants, and the dissociation constants for the binding of peptides to each kinase. The steady-state kinetic mechanism of the reaction catalyzed by the cyclic GMP-dependent protein kinase will also be determined and compared to that of the cyclic AMP-dependent enzyme. Peptides with selective inhibitory activity will be further evaluated as to their ability to inhibit the phosphorylation of endogenous protein substrates in cell-free extracts of tissues rich in smooth muscle. The second section will employ synthetic peptides to investigate the interaction of the regulatory and catalytic subunits of the cyclic AMP-dependent kinase. It is postulated that when a synthetic peptide is bound at the substrate binding site of the catalytic subunit the degree of interaction of this subunit with the regulatory subunit will be decreased. An aryl azide derivative of a substrate peptide will be synthesized and used to photoaffinity label the active site of the enzyme. In a third section, antibodies elicited to model peptide substrates will be used to immunologically characterize sites of phosphorylation in known protein substrates of the cyclic AMP-dependent protein kinase. These studies will provide new information on the biochemistry and substrate specificity of the cyclic nucleotide-dependent protein kinases and may result in new compounds for use as pharmacological tools in the study of the physiological role of these enzymes in hormone action.